Transcriptional repression of VEGF by ZNF24: mechanistic studies and vascular consequences in vivo

Abstract

Key Points• ZNF24 represses VEGF transcription through direct binding to an 11-bp fragment of the VEGF proximal promoter.• ZNF24 functions as a negative regulator of developmental and tumor angiogenesis by inhibiting VEGF transcription.VEGF is a key regulator of normal and pathologic angiogenesis. Although many trans-activating factors of VEGF have been described, the transcriptional repression of VEGF remains much less understood. We have previously reported the identification of a SCAN domain-containing C2H2 zinc finger protein, ZNF24, that represses the transcription of VEGF. In the present study, we identify the mechanism by which ZNF24 represses VEGF transcription. Using reporter gene and electrophoretic mobility shift assays, we identify an 11-bp fragment of the proximal VEGF promoter as the ZNF24-binding site that is essential for ZNF24-mediated repression. We demonstrate in 2 in vivo models the potent inhibitory effect of ZNF24 on the vasculature. Expression of human ZNF24 induced in vivo vascular defects consistent with those induced by VEGF knockdown using a transgenic zebrafish model. These defects could be rescued by VEGF overexpression. Overexpression of ZNF24 in human breast cancer cells also inhibited tumor angiogenesis in an in vivo tumor model. Analyses of human breast cancer tissues showed that ZNF24 and VEGF levels were inversely correlated in malignant compared with normal tissues. These data demonstrate that ZNF24 represses VEGF transcription through direct binding to an 11-bp fragment of the VEGF proximal promoter and that it functions as a negative IntroductionVEGF is a key angiogenic factor during embryogenesis, as demonstrated by the embryonic lethality and abnormal blood vessel development of mice lacking a single VEGF allele. 1 It also plays critical roles in normal physiologic processes such as maintaining vascular stability 2 and promoting skeletal muscle differentiation. 3 Across a wide range of human cancers, VEGF serves as a principle proangiogenic factor. For example, VEGF levels are elevated in breast cancer patients 4 and are inversely correlated with patient survival. 5 Transcriptional regulation of VEGF has been defined in part by its many transcriptional activators, including Sp1, HIF-1, and STAT3. 6,7 In contrast, only the p53 family proteins and the von Hippel-Lindau (VHL) tumor suppressor have been discovered and well characterized as transcriptional repressors of VEGF.Zinc finger protein 24 (ZNF24, also known as ZNF191 or Kox17) was originally identified in an attempt to discover novel C2H2 zinc finger proteins in the hematopoietic system. 8 It possesses 4 Kruppel-like C2H2 zinc finger domains within the c-terminus that function as DNA-binding domains. 9 These domains bind to the TCAT repeats of the HUMTH01 microsatellite located in the first intron of the tyrosine hydroxylase gene in in vitro studies. 9,10 No in vivo function relevant to this observation has been documented to date. The N-terminus of ZNF24 contains a SCAN box domain that primarily serves as a dimerizat...

Prognostic value of vascular endothelial growth factor and hypoxia-inducible factor 1α in canine malignant mammary tumors

“…In accordance with our results, some studies in humans have shown that serum VEGF levels were higher in patients with breast cancer when compared to healthy subjects and inversely correlated with survival (46,47). On the other hand, Duranyildiz et al (48) and El Tarhouny et al (49) did not observe any significant difference between serum VEGF levels in patients with breast cancer and the control group.…”

“…In humans, there was a study that compared the serum levels of HIF-1α in diabetic patients with breast cancer and control groups, demonstrating the HIF-1α levels were markedly higher in the patient group than in the controls (55). Other authors found higher serum HIF-1α levels in patients with lung cancer (57), as well as patients with liver cancer (46).…”

The endogenous zinc finger transcription factor, ZNF24, modulates the angiogenic potential of human microvascular endothelial cells

“…Our results have demonstrated that ZNF24 serves as a new positive regulator of the angiogenic potential of microvascular ECs. Interestingly, we observed the opposite effect of ZNF24 in human breast cancer cells, in which it functions as a potent inhibitor of tumor angiogenesis by repressing the transcription of VEGF (10, 11). One possible reason for the distinct functions of ZNF24 in different cell types is that similar to many other transcription factors, the function of ZNF24 may be context dependent.…”

“…In ECs, ZNF24 regulates the mRNA levels of MMP2, CDKN3 and CCND2. To explore whether MMP2, CDKN3 , and CCND2 are direct target genes for ZNF24 in HMVEC cells, we searched the proximal promoter regions of these genes for known ZNF24 binding sites: TCAT repeats (57), ATTAATT (12), and GCTTTCCATTT (11). There is no known ZNF24 binding site within the proximal promoter regions of these genes, indicating that ZNF24 may regulate the transcription of these genes indirectly.…”

“…Studies in our laboratory have shown that ZNF24 levels are significantly decreased in breast cancer and colon cancer tissues compared to normal tissues. It represses the transcription of VEGF , one of the principal proangiogenic factors, and therefore serves as a potent inhibitor of tumor angiogenesis (10, 11). Conversely, expression of ZNF24 is increased in hepatocellular carcinoma and is positively correlated with the growth of hepatocellular carcinoma cells (12).…”